JP6788376B2 - Method for Producing Peptide Containing Aspartic Acid Residue - Google Patents

Description

The present invention is a method for producing a peptide containing an aspartic acid residue, and a high-purity target peptide can be easily produced by suppressing the production of a specific by-product derived from the aspartic acid residue. It's about the method.

Peptide synthesis methods are roughly classified into solid phase methods and liquid phase methods. In the solid-phase method, it is common to immobilize an amino acid or peptide on a carrier or the like and extend the peptide chain one by one. In the liquid phase method, a peptide fragment consisting of two or more amino acid residues may be bound, but particularly when preparing a peptide fragment, the peptide chains are also extended one by one.

In peptide synthesis, the existence of a wide variety of side reactions in extending a peptide chain is known, and such side reactions can be a major problem in any method. For example, because the physical properties of the by-product produced by the side reaction are similar to those of the target peptide, it may not be finally separated from the target peptide, which may reduce the purity of the target peptide.

Examples of such a side reaction include aspartimization in which the side chain carboxy group of aspartic acid reacts with the α-amino group of an adjacent amino acid residue forming a peptide bond to form a ring structure (Non-Patent Document 1). ).

According to Non-Patent Document 1, aspartimide produced by aspartimization is further attacked by a nucleophilic compound to open the ring, and a new by-product called an aspartimide analog is generated. As a nucleophilic compound, it is used for deprotection of water contained in a solvent and an α-amino group protected by a 9-fluorenylmethyloxycarbonyl group (hereinafter, may be abbreviated as “Fmoc group”). Examples include piperidine. Further, in the aspartic acid ring, if the peptide bond between the α-carboxy group of aspartic acid and the α-amino group of the adjacent amino acid residue is hydrolyzed, the peptide bond is β-isomerized (Patent Document). 1). As a result, the peptide containing an aspartic acid residue is contaminated with not only amino acid deficients, which are common impurities contained in synthetic peptides, but also aspartimide and aspartic acid analogs.

In peptide synthesis, it is common that the target peptide is finally purified by HPLC. However, some aspartimides and analogs of aspartimides are extremely difficult to separate from the target peptide because they have a structure and properties similar to those of the target peptide.

Similarly, in the production of peptides having a structure other than the peptide structure, it is necessary to suppress aspartimization. For example, in the production of lipopeptides, not only the target compound, lipopeptide, but also aspartimide analogs and other by-products show high hydrophobicity under the influence of lipid sites. Since these do not show sharp peaks in general reversed-phase HPLC and elute in similar retention times in low-polarity regions, it is difficult to purify them by HPLC purification as compared with ordinary peptides.

Therefore, prior to purification, a technique has been developed for suppressing the formation of aspartimide and aspartimide analogs in peptide synthesis by devising reaction conditions.

For example, in Patent Document 1, when synthesizing tezglutide, which is a peptide containing aspartic acid residues, aspartic acid is formed by binding two peptide fragments instead of adding amino acid residues one by one. Methods of suppression are disclosed. However, according to the examples of Patent Document 1, each fragment contains as much as tens of percent of aspartimide analogs, and the target peptide, tezglutide, also contains as much as 17% of aspartimide analogs. Patent Document 1 states that "the level of β-Asp analog was significantly reduced" by this method, but this degree of effect is not sufficient at all.

As another technique for suppressing the formation of aspartic acid and aspartic acid analogs, Non-Patent Document 2 describes N- (2-hydroxy-) as an α-amino group contained in a peptide bond on the C side of an aspartic acid residue. A method of protection with a 4-methoxybenzyl) group (Hmb group) is described. Moreover, whereas Patent Document 2, the protecting group of the side chain carboxy group of the aspartic acid residue in the peptide synthesis is generally a t- butyl group (OBu ^t group), 4-(n-propyl ) A method of protecting with a bulky protecting group such as a hepta-4-yl group (OPhp group) or a 3-ethylpenta-3-yl group (Epe group) is disclosed. Further, Non-Patent Document 3 describes a method for removing an Fmoc group in the presence of a base and an organic acid.

International Publication No. 2012/028602 Pamphlet European Patent Application Publication No. 2886531

Ramon Subiros-Funosas et al., Tetrahedron, 67 (2011), 8595-8606 Martin Quibel et al., J. Mol. Chem. Soc. , Chem. Commun. , 1994, 2343-2344 Tillmann Michels et al., Org. Lett. , Vol. 14, No. 20, 2012, 5218-5221

As described above, in the production of peptides containing aspartic acid residues, techniques aimed at suppressing the formation of asparticides and aspartic acid analogs have been developed, but their effects have not been sufficient. For example, in the examples described in Patent Document 1, it is shown that as much as 17% of aspartimide is mixed in the target compound, and in the examples described in Patent Document 2, several% of aspartimide is the target peptide. It is mixed in. Further, even in the experimental data described in Non-Patent Document 3, about 10% or more of aspartimide is mixed in the product.

Some aspartimides and aspartimide analogs have structures and properties similar to those of the target peptide, and it is difficult to separate them from the target peptide even if they are produced, and this tendency is for lipopeptides with relatively high hydrophobicity. It is even more prominent. Therefore, it is necessary to further suppress the production of these by-products.

In the method described in Non-Patent Document 2, the α-amino group of an amino acid must be protected with a bulky protecting group, and the yield of the coupling reaction of such an amino acid is low. Therefore, actual peptide synthesis, particularly It is difficult to apply to industrial peptide synthesis.

Therefore, the present invention is a method for producing a peptide containing an aspartic acid residue, which can remarkably reduce the production of aspartimide and aspartic acid analogs, and can easily produce a high-purity target peptide. The purpose is to provide a method.

As a result of intensive studies to solve the above problems, the present inventors have found a condition that can remarkably suppress the formation of asparticide and aspartic acid analogs in producing a peptide containing an aspartic acid residue, and have found the present invention. Was completed.

Hereinafter, the present invention will be shown.

[1] A method for producing a peptide containing an aspartic acid residue.
A step of introducing an aspartic acid residue by reacting an amino group of the N-terminal or carrier of an intermediate peptide with an aspartic acid derivative represented by the following formula (I).

[In the formula, R ¹ represents a methyl group, R ² and R ³ independently represent a C _2-6 alkyl group or a C _6-10 aryl-C _1-6 alkyl group, R ² and R ³ If indicates a C _2-6 alkyl group, it may be bonded to each other to form a cyclic alkyl group, and Fmoc indicates a 9-fluorenylmethyloxycarbonyl group].
and,
The introduced 9-fluorenylmethyloxycarbonyl group-protected amino group of the aspartic acid residue is based with one or more additives selected from the group consisting of carboxylic acids, sulfonic acids, phenols and acidic alcohols. A method comprising a step of deprotecting and converting to an amino group under basic conditions including.

[2] Since the introduction of the first aspartic acid residue, an amino acid introduction reagent in which the α-amino group is protected with a 9-fluorenylmethyloxycarbonyl group is used and introduced as a reagent for introducing an amino acid. The α-amino group of the amino acid residue is deprotected under a basic condition containing one or more additives selected from the group consisting of carboxylic acids, sulfonic acids, phenols and acidic alcohols, and a base. The method according to [1].

[3] The method according to the above [1] or [2], wherein the additive is a carboxylic acid and / or an acidic alcohol.

[4] The method according to the above [3], wherein the additive is a carboxylic acid.

[5] The method according to the above [4], wherein the additive is formic acid.

[6] The above [1] to [5], wherein the sequence of the above peptide includes one or more sequences selected from the group consisting of Asp-Gly, Asp-Asp, Asp-Asn, Asp-Arg and Asp-Cys. ] The method according to any one of the items.

[7] In any one of the above [1] to [5], the sequence of Ala-Thr-Pro-Glu-Asp-Asn-Gly-Arg-Ser-Phe-Ser is included in the sequence of the peptide. The method described.

[8] The method according to any one of the above [1] to [7], wherein the peptide is a lipopeptide or a glycopeptide.

[9] The peptide has the sequence of Cys-Ala-Thr-Pro-Glu-Asp-Asn-Gly-Arg-Ser-Phe-Ser, and has a side chain thiol group of Cys (2R) -2. , The method according to any one of the above [1] to [8], which is a lipopeptide in which a 3-bis (palmitoyloxy) propyl group is substituted.

In the present invention, the "C _1-6 alkyl group" refers to a linear or branched monovalent saturated aliphatic hydrocarbon group having 1 or more carbon atoms and 6 or less carbon atoms. For example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, n-hexyl and the like. It is preferably a C _1-4 alkyl group, more preferably a C _1-2 alkyl group, and most preferably methyl.

The "C _2-6 alkyl group" refers to the above C _1-6 alkyl group having 2 or more and 6 or less carbon atoms, preferably a C _2-5 alkyl group, and more preferably. It is a C _2-4 alkyl group.

When R ² and R ³ represent a C _2-6 alkyl group and are bonded to each other to form a cyclic alkyl group, examples of such cyclic alkyl group include cyclopentyl, cyclohexyl, adamantyl and the like.

The "C _6-10 aryl group" refers to a monovalent aromatic hydrocarbon group having 6 or more and 10 or less carbon atoms. For example, phenyl, indenyl, naphthyl and the like, preferably phenyl or naphthyl, more preferably phenyl.

According to the method of the present invention, it is possible to remarkably suppress the formation of aspartid and aspartid analogs, which are by-products derived from aspartic acid residues and have a structure and properties close to those of the target peptide and are difficult to separate from the target peptide. As a result, the load in purification is reduced, and a high-purity target peptide can be produced. Further, in the method of the present invention, as an aspartic acid introduction reagent for introducing an aspartic acid residue, a reagent in which the side chain carboxy group is protected by a specific group is used, and the α-amino group is a protecting group. It is very convenient because the conventional peptide synthesis method can be applied as it is, except that a specific reagent is allowed to act for cleavage and removal of the Fmoc group. Therefore, the present invention is extremely useful industrially as a technique capable of easily producing a high-purity peptide.

FIG. 1 is a mass spectrum chart of a crude peptide produced by a conventional method. FIG. 2 is a mass spectrum chart of a crude lipopeptide produced by a conventional method. FIG. 3 is an HPLC chart of a lipopeptide purified by HPLC from a crude lipopeptide produced by a conventional method. FIG. 4 is a mass spectrum chart of a lipopeptide purified by HPLC from a crude lipopeptide produced by a conventional method. FIG. 5 is a mass spectrum chart of the crude peptide produced by the method of the present invention. FIG. 6 is a mass spectrum chart of the crude lipopeptide produced by the method of the present invention. FIG. 7 is a mass spectrum chart of a lipopeptide purified by HPLC from a crude lipopeptide produced by the method of the present invention. FIG. 8 is a mass spectrum chart of a lipopeptide purified by HPLC from a crude lipopeptide produced by the method of the present invention.

In the method of the present invention, a conventional peptide synthesis method can be applied until the aspartic acid residue is introduced. Hereinafter, first, a general peptide synthesis method will be described separately for a coupling reaction and a deprotection reaction for a terminal α-amino group.

1. 1. General Coupling Reaction Step In this step, amino acid residues other than aspartic acid remain by reacting the N-terminal or carrier of the intermediate peptide with derivatives of amino acids other than aspartic acid until the aspartic acid residue is introduced. Introduce the group. Examples of the intermediate peptide include an intermediate peptide for solid phase synthesis in which the C-terminal is immobilized on a carrier, and an intermediate peptide for liquid phase synthesis in which the C-terminal is protected. However, when the C-terminal residue of the target peptide for production is an aspartic acid residue, it is preferable not to carry out this step 1 and the next step 2 but to carry out from step 3 described later.

The carrier of the intermediate peptide for solid-phase synthesis may be any carrier used in general peptide synthesis. Examples of the carrier for solid phase synthesis that can be used in the present invention include Rink amide resin, Rink Amide AM resin, Rink amide PEGA resin, Rink amide MBHA resin, Rink amide MBHA resin, Rink amide AM resin, Rink amide AM resin, and Rink Amide resin. , TentaGel resin, Wang resin, HMPA-PEGA resin, HMP-NovaGel, HMPA-NovaGel, 2-Chlorotrityl resin, NovaSyn TGT alkol resin, HMPB-AM, SeeberSireSiever 3-methoxyphenoxy} butylyl AM resin, 4-Sulfamilybutylyl AM resin, 4-Sulfamilyphenoxy} butylyl AM resin, 4-Fmoc-hydrozinobenzoyl AM resin, 4-Fmoc-hydrozinobenzoyl AM resin, 4-Fmoc-hydrozinobenzoyl AM resin, 4-Fmoc-hydrozinobenzoyl AM resin, 4-Fmoc-hydrozinobenzoyl AM resin, etc.

A carrier for solid-phase synthesis generally has a reactive functional group such as an amino group or a hydroxy group as a starting point for solid-phase synthesis on its surface. When binding the C-terminal amino acid to the carrier, a conventional method may be applied, such as activating the carboxy group of the C-terminal amino acid or the surface functional group of the carrier, or using a coupling reagent. Further, a linker group for binding a C-terminal amino acid may be bonded to the functional group on the surface of the carrier. Further, as the carrier, a carrier to which the C-terminal amino acid of the target peptide or the C-terminal side peptide is originally bound may be used.

The C-terminal protecting group of the intermediate peptide for liquid phase synthesis that can be used in the present invention is not particularly limited as long as it is used in general liquid phase synthesis, but for example, C _1-6 alkyl ester group, C _{3- Examples} thereof include a ₁₀ cycloalkyl ester group, a C _6-13 aryl-C _1-6 alkyl ester group, an amide group and a hydrazide group. Each group may have a substituent. _Examples of the substituent of the C _1-6 alkyl ester group and the C _3-10 cycloalkyl ester group include heteroaryl groups such as a halogeno group, a benzoyl group and a pyridinyl group. _Examples of the substituent of the C _6-13 aryl group include one or more substituents selected from the group consisting of a C _1-6 alkyl group, a C _1-6 alkoxy group, a halogeno group and a nitro group. Examples of the substituent of the amide group and the hydrazide group include a C _1-6 alkyl group, a C _6-13 aryl group, and a C _6-13 aryl-C _1-6 alkyl group. When the amide group and the hydrazide group are substituted with two C _1-6 alkyl groups, they are bonded to each other to form a nitrogen-containing heterocyclyl group such as a pyrrolidinyl group or a piperidinyl group together with the substituted nitrogen atom. May be good.

Specifically, examples of the C _1-6 alkyl ester group include methyl ester, ethyl ester, t-butyl ester, trichloroethyl ester, phenacyl ester, 4-picoryl ester and the like. _{Examples of the} C _3-10 cycloalkyl ester group include cyclohexyl ester and adamantyl ester. _{Examples of the} C _6-13 aryl-C _1-6 alkyl ester group include benzyl ester, p-nitrobenzyl ester, p-methoxybenzidiester, diphenylmethyl ester, and 9-fluorenylmethyl ester. As the amide group, an unsubstituted amide (-(C = O) -NH ₂ ); a primary amide group such as N-methylamide, N-ethylamide, N-benzylamide; N, N-dimethylamide, pyrrolidinylamide And secondary amide groups such as piperidinyl amide. Examples of the hydrazide group include unsubstituted hydrazide (-(C = O) -NHNH ₂ ); substituted hydrazide such as N-phenylhydrazide and N, N'-diisopropylhydrazide.

The amino acid constituting the target peptide produced by the method of the present invention is a natural type as long as it is an amino acid that is a compound having at least one amino group (-NH ₂ ) and one or more carboxy group (-COOH) in addition to aspartic acid. And non-natural type. In the present invention, the amino acid is not particularly limited, but preferably, for example, glycine, alanine, 3,3,3-trifluoroalanine, 2-aminobutanoic acid, 2-amino-2-methylbutanoic acid, nolvalin, etc. 5,5,5-trifluoronorvaline, valine, 2-amino-4-pentenoic acid, 2-amino-2-methylpentenoic acid, propargylglycine, 2-amino-cyclopentanecarboxylic acid, norleucine, leucine, isoleucine, tert-leucine, 2-amino-4-fluoro-4-methylpentanoic acid, 4-aminocyclohexanecarboxylic acid, serine, O-methylserine, O-allylserine, threonine, homoserine, cysteine, 2-methylcysteine, methionine, penicillamine, Asparaginic acid, asparagine, arginine, histidine, glutamic acid, glutamine, 2-aminoadiponic acid, ornithine, tyrosine, tryptophan, lysine, (1R, 2S) -1-amino-2-vinyl-cyclopropanoic acid, (1R, 2S) Α-Amino acids such as -1-amino-2-ethyl-cyclopropanoic acid, sarcosin, phenylalanine, N-methylalanine, N, N-dimethylalanine; aziridinecarboxylic acid, azetidinecarboxylic acid, proline, 1-methylproline, 2-Methylproline, 2-Eethylproline, 3-Methylproline, 4-Methylproline, 5-Methylproline, 4-Methyleneproline, 4-Hydroxyproline, 4-Fluoroproline, pipecholic acid, nipecotic acid, isonipecotic acid, etc. Cyclic amino acids; β-amino acids such as β-alanine, isoselin, 3-aminobutanoic acid, 3-aminopentanoic acid, β-leucine; 4-aminobutanoic acid, 4-amino-3-methylpropionic acid, 4-amino-3-3 Examples thereof include γ-amino acids such as propylbutanoic acid, 4-amino-3-isopropylbutanoic acid and 4-amino-2-hydroxybutanoic acid. Preferred amino acids are natural amino acids and α-amino acids, more preferably natural α-amino acids. In the present invention, the number of amino groups and carboxyl groups in one amino acid molecule is not particularly limited, and for example, 1 It may be one or more. Further, the number of amino groups and carboxyl groups per amino acid molecule may be the same or different.

It is preferable to protect the α-amino group and the side chain reactive functional group of the amino acid introduction reagent used for introducing the C-terminal amino acid into the carrier and for extending the intermediate peptide chain.

Examples of the side chain reactive functional group other than the amino group and the carboxy group required for the coupling reaction in the amino acid introduction reagent include an amino group, a carboxy group, a hydroxyl group, a guanidino group and a mercapto group. These side chain reactive functional groups can be protected with known protecting groups. The protecting group can be appropriately selected depending on the side chain reactive functional group to be protected, and is not particularly limited. For example, a toluenesulfonyl group (Ts group) or tert-butoxy as a protecting group for an amino group. Carbonyl group (Boc group), N- (2-hydroxy-4-methoxybenzyl) group (Hmb group), bis (4-methoxyphenyl) methyl group (Dmb group), benzyloxycarbonyl group, benzyloxymethyl group (Bom) Group), 4,4-dimethyl-2,6-dioxocyclohexylideneail group (Dde group), 1- (4,4-dimethyl-2,6-dioxocyclohexy-1-ylidene) -3- the methylbutyl group (ivDde group); tert- butyl group as a protecting group for a carboxy group (Bu ^t group), O-3- methyl - pent-3-yl group a (Mpe group); a hydroxyl-protecting group tert- butyl group (Bu ^t group); the as a protecting group for the guanidino group 2,2,5,7,8-pentamethylchroman-6-sulfonyl group (Pmc group), 2,2,4,6,7-pentamethyl-dihydro benzofuran-5-sulfonyl group and (Pbf group); trityl group (Trt group) as a protective group of a mercapto group, tert- butyl group (Bu ^t group), N- (acetyl) aminomethyl group (Acm group), trimethyl acetamide Examples thereof include a methyl group (Tacm group), a monomethoxytrityl group (Mmt group), and a 4-methyltrityl group (Mtt group).

It is also preferable to protect the α-amino group of the amino acid introduction reagent. As the protecting group for the α-amino group, a urethane-type protecting group such as a Boc group or an Fmoc group is preferable from the viewpoint of suppressing racemization.

The coupling reaction is preferably carried out in a solvent. The reaction solvent is not particularly limited as long as it does not adversely affect the reaction. For example, ether solvents such as diethyl ether, tetrahydrofuran and 1,4-dioxane; halogenated hydrocarbon solvents such as methylene chloride and dichloroethane; acetonitrile, Nitrogen-containing solvents such as dimethylformamide and dimethylacetamide; alcohol solvents such as methanol and ethanol; dimethyl sulfoxide; water and the like can be used. The reaction solvent may be used alone or in combination of two or more. When two or more kinds of solvents are used, there is no particular limitation on the mixing ratio. As the reaction solvent, dimethylformamide, dimethylacetamide and / or dimethyl sulfoxide are preferably used from the viewpoint of reaction yield.

The amount of the solvent used may be appropriately adjusted, and may be, for example, 0.1% by mass or more and 50% by mass or less with respect to the carrier or intermediate peptide to be subjected to the coupling reaction.

The amount of the amino acid-introducing reagent used for the coupling reaction with the carrier or the intermediate peptide may be appropriately adjusted. For example, 50 times or more the molar amount of the reactive functional group or the intermediate peptide on the carrier is 1 molar or more. The amount can be doubled or less, preferably 1 times or more and 10 times or less.

In the coupling reaction between the carrier or intermediate peptide and the amino acid introduction reagent, it is preferable to activate esterify the carboxy group of the amino acid introduction reagent or use a coupling reagent. Coupling reagents include, for example, triazoles such as 1-hydroxybenzotriazole (HOBt) and 1-hydroxy-7-azabenzotriazole (HOAt); O- (benzotriazole-1-yl) -N, N, N. ', N'-tetramethyluronium hexafluorophosphate (HBTU), O- (7-azabenzotriazole-1-yl) -N, N, N', N'-tetramethyluronium hexafluorophosphate Salt (HATU), O- (6-chlorobenzotriazole-1-yl) -N, N, N', N'-tetramethyluronium hexafluorophosphate (HCTU), N, N, N', N '-Tetramethyl-O- (N-succinimidyl) uronium hexafluorophosphate (TSTU), (1-cyano-2-ethoxy-2-oxoethylideneaminooxy) dimethylaminomorpholinocarbonium hexafluorophosphate (COMU) ), Fluorophosphates such as 1H-benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP); 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC / HCl) ), Carbodiimides such as diisopropylcarbodiimide (DIC) and dicyclohexylcarbodiimide (DCC); propanephosphonic acid anhydride (T3P) and the like.

Further, a base may be added for the purpose of improving the reaction yield. As the base, diisopropylethylamine, triethylamine, N-methylmorpholine and the like can be used.

The reaction conditions may be appropriately adjusted, for example, the reaction temperature may be between the freezing point and the boiling point of the solvent used, but is preferably −20 ° C. or higher, 120 ° C. or lower, and more preferably 0 ° C. or higher. It is 30 ° C. or lower. The reaction time can be, for example, preferably 5 seconds or more and 3 hours or less, preferably 30 seconds or more and 1 hour or less.

The coupling reaction is preferably repeated at least twice until the amino acid to be introduced is sufficiently introduced into the carrier or intermediate peptide. For example, it can be repeated 2 times or more and 10 times or less, preferably 5 times or less.

After completion of the reaction, it is preferable to carry out a usual post-treatment. For example, when the solid-phase method is used, the reaction solution is filtered using a filter having a pore size capable of filtering the carrier, and the filtered carrier is sufficiently washed with a solvent to use an excessive amount of amino acids. Introductory reagents, coupling reagents, etc. can be removed.

The solvent used for washing may be the same as or different from the solvent used for the coupling reaction. The dose of the solvent used for washing and the number of washings may be appropriately adjusted so that the amino acid-introducing reagent and the like can be sufficiently removed. For example, the same amount of solvent as the solvent used for the coupling reaction is used. The number of washings can be 2 or more and 10 or less. When the washing is repeated twice or more, the solvents used may be the same or different.

After washing, it is preferable to dry the carrier. As the drying method, heat drying, vacuum drying, and a combination thereof can be used.

When the liquid phase method is used, it is preferable to add water, an acidic aqueous solution or a basic aqueous solution to the reaction solution after completion of the reaction to stop the reaction, and then perform the extraction operation.

The acidic aqueous solution used for terminating the reaction is not particularly limited as long as it is an aqueous solution containing a general acidic compound. The acidic compound is not particularly limited, and inorganic acidic salts such as alkali metal hydrogen sulfate and dihydrogen phosphate, mineral acids such as hydrochloric acid and sulfuric acid, and organic acids such as citric acid can be used. Of these, acid salts such as potassium hydrogen sulfate and potassium dihydrogen phosphate are preferable.

The basic aqueous solution used for terminating the reaction is not particularly limited as long as it is an aqueous solution containing a general basic compound. The basic compound is not particularly limited, and examples thereof include a basic aqueous solution such as an aqueous solution of sodium hydrogen carbonate, an aqueous solution of sodium carbonate, and an aqueous solution of sodium hydroxide.

The solvent used for extraction is not particularly limited, and for example, aromatic hydrocarbon solvents such as toluene and xylene; halogenated hydrocarbon solvents such as dichloromethane, chloroform, 1,2-dichloroethane and chlorobenzene; tetrahydrofuran, 1,4 Ether-based solvents such as -dioxane, t-butylmethyl ether and diisopropyl ether; fatty acid ester-based solvents such as methyl acetate, ethyl acetate and isopropyl acetate; N, N-di-n-propylformamide, N, N-dibutylformamide ( Examples thereof include an aprotic polar solvent that is immiscible with water such as DBF). These solvents may be used alone or in combination of two or more. When two or more kinds are mixed and used, the mixing ratio is not particularly limited.

The obtained extract may be washed with water, an acidic aqueous solution or a basic aqueous solution. As the acidic aqueous solution or the basic aqueous solution, those listed above can be used. When cleaning the extract, in order to make it easier to separate into two layers, an organic layer and an aqueous layer, treatments performed by general extraction operations such as adding inorganic salts such as sodium chloride and sodium sulfate are performed. You may.

The extracted extract after washing may be neutralized if necessary, or may be further washed with saturated brine. Further, the extract may be dried over anhydrous magnesium sulfate or anhydrous sodium anhydride.

The residue obtained by distilling off the reaction solvent and the extraction solvent from the extract obtained in this manner by an operation such as heating under reduced pressure may be used as it is in the next step, or salt formation crystallization or column chromatography. The purity may be further increased by a general purification method such as.

Next, the deprotection reaction of the terminal α-amino group in general peptide synthesis will be described.

2. 2. General Deprotection Reaction Steps for Terminal α-Amino Acids The α-amino group of the N-terminal amino acid of an amino acid coupled to a carrier or intermediate peptide is generally protected by a protecting group. By deprotecting the α-amino acid, further coupling of amino acid residues becomes possible. In this step, the α-amino group is deprotected.

Deprotection of the terminal α-amino group can be carried out by a person skilled in the art by a known method depending on the type of protecting group. For example, in the case of an Fmoc group, a base may be allowed to act on the intermediate peptide in the solvent.

The solvent used in this step is not particularly limited as long as it does not adversely affect the deprotection reaction, and is, for example, an ether solvent such as diethyl ether, tetrahydrofuran or 1,4-dioxane; a halogenated hydrocarbon solvent such as methylene chloride or dichloroethane. Nitrogen-containing solvents such as acetonitrile, dimethylformamide and dimethylacetamide; alcohol-based solvents such as methanol and ethanol; dimethyl sulfoxide; water can be used. These may be used alone or in combination of two or more. When two or more kinds of solvents are used, the mixing ratio is not particularly limited. From the viewpoint of reaction yield, dimethylformamide, dimethylacetamide and / or dimethyl sulfoxide are preferable. As the amount of the solvent used, for example, 0.1 mass times or more and 50 mass times or less may be used with respect to 1 mass times of the intermediate peptide.

For example, as the base for cleaving and removing the Fmoc group, piperidine, piperidine, tetrabutylammonium fluoride, diethylamine, dicyclohexylamine, morpholine, tris (2-aminoethyl) amine, p-dimethylaminopyridine, triethylamine, diisopropyl1, Examples thereof include 8-diazabicyclo [5.4.0] -7-undecene, potassium fluoride, and aluminum chloride. The amount of the base used can be, for example, 1-fold mol or more and 1000-fold mol or less, preferably 1-fold mol or more and 100-fold mol or less, relative to 1-fold mol of the intermediate peptide.

For example, the reaction conditions for cleaving and removing the Fmoc group may be appropriately adjusted. For example, the reaction temperature may be between the freezing point and the boiling point of the solvent used, preferably −20 ° C. or higher and 120 ° C. or lower, and more preferably 20 ° C. or higher and 100 ° C. or lower. The reaction time is preferably 5 seconds or more and 3 hours or less, and more preferably 10 seconds or more and 1 hour or less.

Additives may be used to cleave and remove the Fmoc group. Examples of the additive include 1-octanethiol.

After the deprotection reaction, it is preferable to purify the intermediate peptide by removing the deprotecting reagent and the like by filtering and washing the intermediate peptide as in the treatment after the coupling reaction.

The above deprotection reaction and post-treatment are preferably repeated two or more times until the N-terminal α-amino group can be completely deprotected. For example, it can be repeated 2 times or more and 10 times or less, preferably 5 times or less.

In the method of the present invention, the peptide is obtained by repeating the above-mentioned coupling reaction step 1 and the deprotection reaction step 2 of the terminal α-amino group, if necessary, until the aspartic acid residue is first introduced into the carrier or intermediate peptide. Stretch the chain to synthesize an intermediate peptide.

3. 3. Coupling reaction step of carrier or intermediate peptide and aspartic acid introduction reagent In the present invention, the N-terminal amino group of the intermediate peptide produced above or the amino group of the carrier and α- of the aspartic acid derivative (I) Aspartic acid residues are introduced by reacting with a carboxy group.

In the conventional peptide synthesis method, when aspartic acid is introduced, the side chain carboxy group is isolated on the nitrogen atom in the peptide bond formed between the α-carboxy group of aspartic acid and the adjacent amino acid or carrier. The electron pair reacts and aspartimide tends to be produced as a by-product. In the present invention, along with the characteristic deprotection reaction described later, the α-amino acid protecting group is protected by an Fmoc group and the side chain carboxy group is protected by a specific protecting group as a reagent for introducing aspartic acid. By using the aspartic acid derivative (I), aspartic acid and its relatives by-products are remarkably suppressed.

However, in the present invention, it is not always necessary to use the aspartic acid derivative (I) for each introduction of aspartic acid residue as long as the formation of aspartic acid or aspartic acid analog can be remarkably suppressed. For example, the aspartic acid derivative (I) may be used only at a position where aspartic acid or an aspartic acid analog is likely to be formed, and an aspartic acid introduction reagent other than the aspartic acid derivative (I) may be used at other positions according to the above step 1. ..

This step can be carried out in the same manner as in the coupling step 1 except that the aspartic acid derivative (I) is used as the amino acid introduction reagent.

4. Deprotection reaction step of α-amino group of introduced aspartic acid residue In this step, α- of the aspartic acid residue introduced in the coupling reaction step 3 of the above carrier or intermediate peptide and aspartic acid derivative (I) Deprotects the amino group. However, in such deprotection, in addition to the basic conditions containing a base, which is the usual Fmoc group cleavage reaction condition, one or more additives selected from the group consisting of carboxylic acids, sulfonic acids, phenols and acidic alcohols. It is preferable to use in combination. In the method of the present invention, in addition to using the aspartic acid derivative (I) as the aspartic acid-introducing reagent, the Fmoc group thereof is cleaved and removed under special conditions to significantly contaminate the target peptide with aspartic acid and its analogs. It becomes possible to suppress.

The carboxylic acid is not particularly limited, but is preferably, for example, formic acid, acetic acid, propionic acid, butanoic acid, benzoic acid, salicylic acid, trifluoroacetic acid, difluoroacetic acid, fluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and chloro. Acetic acid and the like can be mentioned, and formic acid is preferable.

The sulfonic acid is not particularly limited, but preferably, for example, sulfuric acid, methanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, trifluoromethanesulfonic acid, taurine and the like can be mentioned.

The above-mentioned phenols are not particularly limited, but preferred examples thereof include phenol, 4-nitrophenol, 3-nitrophenol, 2-nitrophenol, 2,4-dinitrophenol and the like.

The acidic alcohols are not particularly limited, but preferred examples thereof include hexafluoroisopropyl alcohol (HFIP), 2,2,2-trifluoroethanol, and ascorbic acid.

The amount of the additive used may be appropriately adjusted within a range in which the formation of aspartimide and its analogs is suppressed, and is not particularly limited. For example, with respect to 1 mass times the reaction solution used for the deprotection reaction. It can be 0.001 times by mass or more and 0.5 mass times or less, preferably 0.01 mass times or more and 0.1 mass times or less.

Other deprotection reaction conditions and post-treatment conditions may be the same as those in the above-mentioned deprotection reaction step 2 of the terminal α-amino group.

5. Peptide chain extension step After the initial introduction of an aspartic acid residue into a carrier or intermediate peptide, the α-amino group is used as an amino acid introduction reagent when introducing not only aspartic acid but also other amino acid residues. It is preferable to use a peptide protected with an Fmoc group and to deprotect the α-amino group under the conditions of the deprotection reaction step 4, that is, basic conditions containing one or more additives and a base. .. Thereby, it becomes possible to suppress the production of aspartimide and its analogs even more remarkably.

6. Other Reaction Steps In the present invention, the "peptide" has a structure in which two or more amino acids are bound by a peptide bond, and as long as it has the peptide structure, it also includes those having other structures. The peptide having a structure other than the peptide structure is not particularly limited, and examples thereof include a lipopeptide having a lipid moiety such as a long-chain hydrocarbyl group and a glycopeptide to which a sugar is bound.

Examples of the lipid contained in the lipopeptide include one or more long-chain hydrocarbyls. The long-chain hydrocarbyl group refers to a linear alkyl group or alkenyl group having at least 5 carbon atoms, and examples thereof include a C _8-50 alkyl group or a C _8-50 alkenyl group, preferably C _{8- It is a 25} alkyl group or a C _8-25 alkenyl group. Alkenyl groups preferably have one, two or three double bonds, each with an E or Z geometry within the hydrocarbon chain, as is commonly found in natural fatty acids and aliphatic alcohols. .. The definition of long-chain hydrocarbyl further has a branched alkyl or alkenyl group, eg, a methyl or ethyl group at the second or third carbon atom counting from the end of the chain, as in the case of 2-ethylhexyl. Alkyl groups and alkenyl groups are also included.

The number of long-chain hydrocarbyl groups contained in the lipopeptide may be one or two or more. The number is preferably 1 or more and 5 or less.

Examples of the lipid portion of the lipopeptide include S-glycerylcysteine containing 2 or 3 hydrocarbyl groups and a phospholipid containing 2 or 3 hydrocarbyl groups.

The sugar contained in the glycopeptide is not particularly limited, and may be a monosaccharide or a polysaccharide in which two or more sugars are glycosidic bonded. For example, a class of sugars such as pentose, deoxypentose, hexose, deoxyhexose; specific sugars such as glucose, arabinose, xylose, lyxose; sugar analogs such as cyclopentyl groups; and two or more polymers of these. Can be mentioned. The sugar can be in pyranosyl or furanosyl form.

When the peptide has a structure other than the peptide structure, the structure may be directly bound to the peptide structure or may be bound via a linker group. The linker group is not particularly limited, but is, for example, a C _1-6 alkylene group, an amino group (-NH-), an imino group (> C = N- or -N = C <), and an ether group. (-O-), thioether group (-S-), carbonyl group (-C (= O)-), thionyl group (-C (= S)-), ester group (-C (= O) -O-) Or -OC (= O)-), amide group (-C (= O) -NH- or -NH-C (= O)-), sulfoxide group (-S (= O)-), sulfonyl group (-S (= O) _2- ), sulfonylamide groups (-NH-S (= O) _2- and -S (= O) _2- NH-), and groups to which these two or more are bound can be mentioned. it can. When two or more of these groups are bonded to form the linker group, the number of bonds is preferably 10 or less or 5 or less, and more preferably 3 or less.

In this step, if necessary, a structure other than the peptide is bound to the intermediate peptide. For those skilled in the art, it is easy to attach a structure other than the peptide structure directly to the peptide structure or to attach it via a linker group.

Since the intermediate peptide to which a structure other than the peptide structure is bound is bound to the carrier, it can be purified by washing in the same manner as in the above-mentioned coupling reaction step 1.

7. Side chain deprotection and excision from carrier The intermediate peptide synthesized above is intended by deprotecting the reactive functional groups of the side chain by a known method, cleaving from the carrier, and then purifying by washing. It can be a peptide. Aspartic acid and its analogs, which are by-products associated with the introduction of aspartic acid residues, are generally very difficult to separate from the target peptide because they are similar in structure and properties to the target peptide. Since the formation of aspartimide and its analogs is remarkably suppressed, it can be said that the purity of the target peptide produced by the method of the present invention is extremely high.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited by the following examples as well as the present invention, and appropriate modifications are made to the extent that it can be adapted to the gist of the above and the following. Of course, it is possible to carry out, and all of them are included in the technical scope of the present invention.

Comparative Example 1: Synthesis of lipopeptide by general solid-phase peptide synthesis (1) Synthesis of APEDNGRSFS with carrier

(1-1) Coupling reaction between carrier and FmocPhe-OH FmocPhe-OH (397 mg, 1.03 mmol), O- (1H-benzotriazole-1-yl) -N, N, N', N'-tetra Methyluronium hexafluorophosphate (389 mg, 1.03 mmol, hereinafter abbreviated as "HBTU"), 1-hydroxy-1H-benzotriazole (139 mg, 1.03 mmol, hereinafter abbreviated as "HOBT") and Ethyldiisopropylamine (0.196 mL, 2.05 mmol) was added to a suspension consisting of dimethylformamide (3 mL, hereinafter abbreviated as "DMF") and treated with ultrasonic waves for 1 minute to prepare a solution. This solution was added to H-Ser (tBu) -2-chlorotrityl resin (1.14 mmol / g, 300 mg, 0.34 mmol) and stirred at 25 ° C. for 20 minutes using a vortex mixer. The reaction mixture was filtered, and the filtered resin carrier was washed 3 times with DMF (3 mL) and 1 time with dichloromethane (3 mL). After performing the above coupling reaction three times in total, the completion of the reaction was confirmed by the Kaiser test.

(1-2) Deprotection reaction of amino group A 20% piperidine / DMF solution (3 mL) was added to the above resin carrier, and the mixture was stirred at 25 ° C. for 20 minutes using a vortex mixer. The reaction mixture was filtered, and the filtered resin was washed 3 times with DMF (3 mL) and 1 time with dichloromethane (3 mL). By carrying out the above deprotection reaction a total of 4 times, the Fmoc group was removed and the amino group was deprotected.

(1-3) Stretching of peptide chain The coupling reaction of (1-1) above and the deprotection reaction of (1-2) above are repeated, and 1.0698 g of carrier with side chain functional groups protected as described above is attached. Obtained APPEDNGRSFS. Assuming the purity of the obtained product was 100%, the yield was almost 100%.

(1-4) Acquisition of crude ATPEDNGRSFS Trifluoroacetic acid / water / triisopropylsilane = 95 / 2.5 / 2.5 in the side chain protection ATPEDNGRSFS with carrier (0.320 mmol / mg, 30 mg, 0.00953 mmol). A mixture of (vol / vol / vol) (1 mL) was added and stirred at 25 ° C. for 2 hours using a vortex mixer. The reaction solution was filtered, and the filtered resin carrier was washed with trifluoroacetic acid (1 mL, hereinafter abbreviated as "TFA"). The obtained filtrate and washing solution were washed with hexane (5 mL), and then TFA was distilled off under reduced pressure. When diethyl ether (5 mL) was added thereto, a white suspension was formed. After centrifugation, the supernatant was removed and the precipitate was washed 3 times with diethyl ether (5 mL). The precipitate was dried under reduced pressure to obtain crude APPEDNGRSFS as a 9.3 mg white powder (0.00788 mmol, yield 83%, assuming 100% purity).

(1-5) Analysis of crude ATPEDNGRSFS When the powder obtained in (1-4) above was analyzed by MALDI-TOF MS, a plurality of by-products were detected in addition to the target compound ATPEDNGRSFS. From the molecular weight, it was determined that the by-products included aspartimide and aspartimide analogs. The sum of the intensities of the detected signals was set to 100, and the ratio of the intensities of each compound was calculated as the content rate (purity). The mass spectrum chart is shown in FIG. 1, and the analysis results are shown in Table 1.

As shown in the above results, the purity of ATPEDNGRSFS is only 62%, and aspartimide (11%) and aspartimide analog (27%) are contained in a total of 38%, and many impurities having a chemical structure similar to that of the target compound are generated. It turned out that it had been done.

(2) Synthesis of lipopeptide with carrier

(2-1) (R) -Pam2Cys introduction reaction Dichloromethane / N-methylpyrrolidone = 1/1 (vol) was added to the carrier-attached side chain-protected ATPEDNGRSFS (0.32 mmol / g, 200 mg, 0.064 mmol) obtained above. / Vol) mixed solvent (1.0 mL), Fmoc-protected S-[(2R) -2,3-bis (palmitoyloxy) propyl] cysteine (114 mg, 0.128 mmol, hereinafter "(R)-" (Abbreviated as "Pam2Cys"), hexafluorophosphoric acid (benzotriazole-1-yloxy) tripyrolidinophosphonium (66.6 mg, 0.128 mmol, hereinafter abbreviated as "PyBOP") and ethyldiisopropylamine (12.3 μL, 0.0704 mmol) was added in order, and the mixture was stirred at 25 ° C. for 30 minutes using a vortex mixer. Ethyldiisopropylamine (10.1 μL, 0.0576 mmol) was added, and the mixture was further stirred for 2 hours. The reaction mixture was filtered, and the filtered resin carrier was washed 3 times with DMF (3 mL) and 1 time with dichloromethane (3 mL). The Kaiser test confirmed that the reaction was complete.

(2-2) Deprotection reaction of amino group A 20% piperidine / DMF solution (2 mL) was added to the above resin carrier, and the mixture was stirred at 25 ° C. for 20 minutes using a vortex mixer. The reaction mixture was filtered, and the filtered resin carrier was washed 3 times with DMF (3 mL) and 1 time with dichloromethane (3 mL). By carrying out the above deprotection reaction a total of 4 times, the Fmoc group was removed and the amino group was deprotected.

(2-3) Deprotection of side chain functional group and cleavage of carrier Trifluoroacetic acid / in the side chain protecting peptide with carrier (0.064 mmol assuming 100% purity) obtained in (2-2) above. Add a mixture (4 mL) of phenol / water / methylthiobenzene / 3,6-dioxa-1,8-octanedithiol = 79/8/5/5/3 (vol / vol) and use a vortex mixer at 25 ° C. for 2 Stirred for hours. The reaction solution was filtered, and the filtered resin carrier was washed with TFA (1 mL). The obtained filtrate and washing solution were washed with hexane (10 mL), and then TFA was distilled off under reduced pressure. When diethyl ether (10 mL) was added thereto, a white suspension was formed. After centrifugation, the supernatant was removed and the precipitate was washed 3 times with diethyl ether (10 mL). The precipitate was dried under reduced pressure to obtain 79 mg of a white powder (0.0431 mmol, yield 67%, assuming 100% purity).

(2-4) Analysis of crude lipopeptide When the powder obtained in (2-3) above was analyzed by MALDI-TOF MS, a plurality of by-products were detected in addition to the target compound lipopeptide. From the molecular weight, it was determined that the by-products included aspartimide and aspartimide analogs. The sum of the intensities of the detected signals was set to 100, and the ratio of the intensities of each compound was calculated as the content rate (purity). The mass chart is shown in FIG. 2, and the analysis results are shown in Table 2.

As shown in the above results, the purity of the lipopeptide, which is the target compound, is only 51%, and aspartimide (22%) and aspartimide analog (27%) are contained in a total of 49%, which are similar in chemical structure to the target compound. It was found that a lot of impurities were generated.

(2-5) Purification of Lipopeptide From the crude lipopeptide obtained in (2-3) above, the lipopeptide, which is the target compound, was purified by high performance liquid chromatography (HPLC) under the following conditions, and the purified lipo was purified. Peptides were analyzed by HPLC. The conditions for performing each HPLC are shown below.

[HPLC purification conditions]
Column: YMC C4colum (250 x 20 mm)
Eluent: 0.1% TFA aqueous solution / 0.1% acetonitrile solution = 100/0 → 0/100 (0 to 20 min) and 0/100 (20 to 40 min)
Flow velocity: 20 mL / min
Detection wavelength: 220 nm
Elution time: 26 min

[HPLC conditions for analysis of purification results]
HPLC column: YMC C4column (250 x 4.7 mm)
Flow velocity: 1.0 mL / min
Column temperature: 40 ° C
Detection wavelength: 220 nm
Eluent: 0.1% TFA aqueous solution / 0.1% acetonitrile solution = 100/0 → 0/100 (0 to 20 min)
Retention time: 17.0 min
Area ratio: 100%
The HPLC chart of the purified lipopeptide is shown in FIG. As shown in FIG. 3, only one peak was obtained (area ratio 100%).

(2-6) Analysis of purified lipopeptide The purified lipopeptide obtained in (2-5) above was analyzed by MALDI-TOF MS, and the content of each compound was calculated in the same manner as in (2-4) above. The mass chart is shown in FIG. 4, and the analysis results are shown in Table 3.

As shown in the above results, impurities such as aspartimide analogs could be removed by HPLC, but aspartimide could not be removed (38%), and the purity of lipopeptide was only improved to 62% regardless of purification by HPLC.

Example 1: Synthesis of lipopeptide by the method of the present invention (1) Synthesis of side chain functional group-protected NGRSFS with a carrier

(1-1) Coupling reaction between the carrier and FmocPhe-OH In the same manner as in Comparative Example 1 above, the carrier and FmocPhe-OH were coupled under general conditions. Specifically, ethyl is added to a suspension consisting of FmocPhe-OH (1.19 g, 3.08 mmol), HBTU (1.17 g, 3.08 mmol), HOBT (416 mg, 3.08 mmol) and DMF (9 mL). Diisopropylamine (0.589 mL, 6.16 mmol) was added and sonicated for 1 minute to prepare a solution. This solution was added to H-Ser (tBu) -2-chlorotrityl resin (1.14 mmol / g, 900 mg, 1.03 mmol) and stirred at 25 ° C. for 20 minutes using a vortex mixer. The reaction mixture was filtered, and the filtered resin carrier was washed 3 times with DMF (9 mL) and 1 time with dichloromethane (9 mL). After performing the above coupling reaction three times in total, the completion of the reaction was confirmed by the Kaiser test.

(1-2) Deprotection reaction of amino group A 20% piperidine / DMF solution (9 mL) was added to the above resin carrier, and the mixture was stirred at 25 ° C. for 20 minutes using a vortex mixer. The reaction mixture was filtered, and the filtered resin carrier was washed 3 times with DMF (9 mL) and 1 time with dichloromethane (9 mL). By carrying out the above deprotection reaction a total of 4 times, the Fmoc group was removed and the amino group was deprotected.

(1-3) Stretching of peptide chain The coupling reaction of (1-1) above and the deprotection reaction of (1-2) above were repeated, with 2.25 g of carrier in which the side chain functional group was protected as described above. Obtained side chain functional group protection NGRSFS. Assuming the purity of the obtained product was 100%, the yield was almost 100%.

(2) Synthesis of side chain functional group-protected DNGRSFS with carrier

(2-1) Coupling of D with side chain carboxy group protected with MPe and side chain functional group protected NGRSFS with carrier Fmoc-Asp (OMpe) -OH (395.6 mg, 0.9 mmol) and HBTU (341) Ethyldiisopropylamine (0.314 mL, 1.8 mmol) was added to a mixture consisting of .4 mg, 0.9 mmol), HOBT (121.6 mg, 0.9 mmol), and DMF (3 mL), and the mixture was subjected to ultrasonic treatment for 1 minute. , As a solution. This was added to side chain functional group protected NGRSPS with a carrier (0.459 mmol / g; 654 mg, 0.3 mmol), and the mixture was stirred at 25 ° C. for 30 minutes using a vortex mixer. The reaction mixture was filtered, and the resin carrier was washed 3 times with DMF (3 mL) and 1 time with dichloromethane (3 mL), and then dried under reduced pressure. After performing the above coupling operation, the completion of the reaction was confirmed by the Kaiser test.

(2-2) Deprotection Reaction of Amino Group A mixed solution (3 mL) of 1% formic acid / 19% piperidine / 80% DMF was added to the above resin carrier, and the mixture was stirred at 25 ° C. for 20 minutes using a vortex mixer. The reaction mixture was filtered, and the filtered resin carrier was washed 3 times with DMF (3 mL) and 1 time with dichloromethane (3 mL). By carrying out the above deprotection reaction a total of 4 times, the Fmoc group was removed to obtain a carrier-attached side chain functional group-protected DNGRSFS.

(3) Synthesis of side chain functional group-protected APEDNGRSFS with carrier

(3-1) Coupling of Fmoc-protected amino acid and intermediate peptide As a typical example, the reaction between carrier-attached side chain functional group-protected DNGRSFS and FmocGlu (tBu) -OH is described. Ethyldiisopropylamine (0.172 mL) in a suspension consisting of FmocGlu (tBu) -OH (383 mg, 0.9 mmol), HBTU (341 mg, 0.9 mmol), HOBT (122 mg, 0.9 mmol) and DMF (3 mL). , 1.8 mmol) was added and sonicated for 1 minute to prepare a solution. This solution was added to the carrier-attached side chain functional group-protected DNGRSFS (0.3 mmol) obtained in (2) above, and stirred at 25 ° C. for 20 minutes using a vortex mixer. The reaction mixture was filtered, and the filtered resin carrier was washed 3 times with DMF (3 mL) and 1 time with dichloromethane (3 mL). After performing the above coupling operation a total of 3 times, the completion of the reaction was confirmed by the Kaiser test.

(3-2) Deprotection reaction of amino group A mixed solution (3 mL) of 1% formic acid / 19% piperidine / 80% DMF was added to the above resin carrier, and the mixture was stirred at 25 ° C. for 20 minutes using a vortex mixer. The reaction mixture was filtered, and the filtered resin carrier was washed 3 times with DMF (3 mL) and 1 time with dichloromethane (3 mL). The Fmoc group was removed by carrying out the above deprotection reaction a total of 4 times.

(3-3) Stretching of peptide chain The coupling reaction of (3-1) above and the deprotection reaction of (3-2) above were repeated, and 0.896 g of carrier with side chain functional groups protected as described above was added. Obtained ATPEDNGRSFS. Assuming the purity of the obtained product was 100%, the yield was approximately 100% and the number of moles per gram was 0.335 mmol / g.

(3-4) Acquisition of crude ATPEDNGRSFS TFA / water / triisopropylsilane = 95 / in the carrier-attached side chain-protected ATPEDNGRSFS (0.335 mmol / mg, 20 mg, 0.00670 mmol) obtained in (3-3) above. A 2.5 / 2.5 (vol / vol / vol) mixture (1 mL) was added and stirred at 25 ° C. for 2 hours using a vortex mixer. The reaction solution was filtered, and the filtered resin carrier was washed with TFA (1 mL). The obtained filtrate and washing solution were washed with hexane (5 mL), and then TFA was distilled off under reduced pressure. When diethyl ether (5 mL) was added thereto, a white suspension was formed. After centrifugation, the supernatant was removed and the precipitate was washed 3 times with diethyl ether (5 mL). The precipitate was dried under reduced pressure to obtain 7.0 mg of crude APEDNGRSFS as a white powder (0.00593 mmol, yield 88%, assuming 100% purity).

(3-5) Analysis of crude ATPEDNGRSFS When the powder obtained in (3-4) above was analyzed by MALDI-TOF MS, a plurality of by-products were detected in addition to the target compound ATPEDNGRSFS. From the molecular weight, it was determined that the by-products included aspartimide and aspartimide analogs. The sum of the intensities of the detected signals was set to 100, and the ratio of the intensities of each compound was calculated as the content rate (purity). The mass spectrum chart is shown in FIG. 5, and the analysis results are shown in Table 4.

As shown in the above results, it was found that the formation of aspartic acid and aspartic acid analogs was suppressed by protecting the side chain carboxy group of aspartic acid with MPe.

(4) Synthesis of lipopeptide with carrier

(4-1) (R) -Pam2Cys introduction reaction Dichloromethane / N-methylpyrrolidone = 1/1 (vol) was added to the carrier-attached side chain-protected ATPEDNGRSFS (0.335 mmol / g, 200 mg, 0.067 mmol) obtained above. / Vol) mixed solvent (1.0 mL), Fmoc-protected (R) -Pam2Cys (120 mg, 0.134 mmol), PyBOP (69.7 mg, 0.134 mmol) and ethyldiisopropylamine (12.9 μL, 0). .0737 mmol) was added in order, and the mixture was stirred at 25 ° C. for 30 minutes using a vortex mixer. Ethyldiisopropylamine (10.5 μL, 0.0603 mmol) was added, and the mixture was further stirred for 2 hours. The reaction mixture was filtered, and the filtered resin carrier was washed 3 times with DMF (3 mL) and 1 time with dichloromethane (3 mL). The Kaiser test confirmed that the reaction was complete.

(4-2) Deprotection reaction of amino group in (R) -Pam2Cys A mixed solution (2 mL) of 1% formic acid / 19% piperidine / 80% DMF was added to the above resin carrier, and 20 at 25 ° C. using a vortex mixer. Stir for minutes. The reaction mixture was filtered, and the filtered resin was washed 3 times with DMF (3 mL) and 1 time with dichloromethane (3 mL). By carrying out the above deprotection reaction a total of 4 times, the Fmoc group was removed and the amino group was deprotected.

(4-3) Deprotection of side chain functional group and cleavage of carrier Trifluoroacetic acid was added to the side chain protected lipopeptide with carrier (0.067 mmol assuming 100% purity) obtained in (4-2) above. Add a mixture (2 mL) of / phenol / water / methylthiobenzene / 3,6-dioxa-1,8-octanedithiol = 79/8/5/5/3 (vol / vol) and use a vortex mixer at 25 ° C. The mixture was stirred for 4 hours. The reaction solution was filtered, and the filtered resin carrier was washed with TFA (1 mL). The obtained filtrate and washing solution were washed with hexane (10 mL), and then TFA was distilled off under reduced pressure. When diethyl ether (10 mL) was added thereto, a white suspension was formed. After centrifugation, the supernatant was removed and the precipitate was washed 3 times with diethyl ether (10 mL). The precipitate was dried under reduced pressure to obtain 79 mg of crude lipopeptide as a white powder (yield 64% assuming 100% purity).

(4-4) Analysis of crude lipopeptide When the powder obtained in (4-3) above was analyzed by MALDI-TOF MS, cysteine deficiency and palmitoyl group deficiency were confirmed in addition to the target compound lipopeptide. On the other hand, aspartimide and aspartimide analogs were not detected. The sum of the intensities of the detected signals was set to 100, and the ratio of the intensities of each compound was calculated as the content rate (purity). The mass chart is shown in FIG. 6, and the analysis results are shown in Table 5.

As shown in the above results, it was found that the formation of aspartic acid and aspartic acid analogs was suppressed by protecting the side chain carboxy group of aspartic acid with MPe.

(4-5) Purification of Lipopeptide From the crude lipopeptide obtained in (4-3) above, the lipopeptide which is the target compound is purified by high performance liquid chromatography (HPLC) under the following conditions, and the purified lipo is purified. Peptides were analyzed by HPLC. The conditions for performing each HPLC are shown below.

[HPLC purification conditions]
Column: YMC C4colum (250 x 20 mm)
Eluent: 0.1% TFA aqueous solution / 0.1% acetonitrile solution = 100/0 → 70/30 (0 to 5 min) and 70/30 → 40/60 (5 to 40 min)
Flow velocity: 20 mL / min
Detection wavelength: 220 nm
Elution time: 38 min

[HPLC conditions for analysis of purification results]
HPLC column: YMC C4column (250 x 4.7 mm)
Flow velocity: 1.0 mL / min
Column temperature: 40 ° C
Detection wavelength: 220 nm
Eluent: 0.1% TFA aqueous solution / 0.1% acetonitrile solution = 100/0 → 70/30 (0 to 5 min) and 70/30 → 40/60 (5 to 40 min)
Holding time: 25.8 min
Area ratio: 100%
An HPLC chart of the purified lipopeptide is shown in FIG. As shown in FIG. 7, only one peak was obtained (area ratio 100%).

(4-6) Analysis of purified lipopeptide The purified lipopeptide obtained in (4-5) above was analyzed by MALDI-TOF MS, and the content of each compound was calculated in the same manner as in (4-4) above. The mass chart is shown in FIG. 8, and the analysis results are shown in Table 6.

As shown in the above results, no by-products with similar structures have been produced, and the lipopeptide, which is the target compound, can be separated from other by-products by HPLC, and its purity is as high as 100%. there were.

As shown in the above results, in the solid phase synthesis of peptides, the side chain carboxy group is protected by O-3-methyl-pent-3-yl (Mpe) as an amino acid introduction reagent for the introduction of aspartic acid residues. In addition, aspartic acid in which the amino group is protected by Fmoc is used, and after the introduction of the aspartic acid residue, an amino acid introduction reagent in which the amino group is protected by Fmoc is used as the amino acid introduction reagent for introducing the amino acid residue, and the Fmoc is used. It has been clarified that by removing the group under basic conditions containing a secondary amine and a specific additive, the formation of aspartimide and its relatives, which are impurities peculiar to peptides containing aspartic acid residues, can be significantly suppressed. It was.

Claims (9)

A method for producing peptides containing aspartic acid residues.
A step of introducing an aspartic acid residue by reacting an amino group of the N-terminal or carrier of an intermediate peptide with an aspartic acid derivative represented by the following formula (I).

[Shown in the formula, the R ¹ is a methyl group, R ² and R ³ independently represent a C _2-6 alkyl group, and R ² and R ³ are bonded to the physician each other to form a cyclic alkyl group Fmoc exhibits a 9-fluorenylmethyloxycarbonyl group]
and,
The step of deprotecting the 9-fluorenylmethyloxycarbonyl group-protected amino group of the introduced aspartic acid residue under basic conditions containing a carboxylic acid and a base and converting it into an amino group is included. How to feature. Since the introduction of the first aspartic acid residue, an amino acid introduction reagent in which the α-amino group is protected by a 9-fluorenylmethyloxycarbonyl group is used as a reagent for introducing an amino acid, and the introduced amino acid is used. The method according to claim 1, wherein the α-amino group of the residue is deprotected under a basic condition containing a carboxylic acid and a base. The amount of the carboxylic acid used is 0.001 with respect to 1 mass times the reaction solution in the step of deprotecting the 9-fluorenylmethyloxycarbonyl group-protected amino group of the aspartic acid residue and converting it into an amino group. The method according to claim 1 or 2, wherein the mass is multiplied by 5 or more and 0.5 mass times or less . The method according to claim 3, wherein the amount of the base used is 1-fold or more and 1000-fold or less with respect to 1-fold molar amount of the intermediate peptide into which the aspartic acid residue has been introduced . The method according to claim 4, wherein the carboxylic acid is formic acid . Any one of claims 1 to 5, wherein the sequence of the peptide includes one or more sequences selected from the group consisting of Asp-Gly, Asp-Asp, Asp-Asn, Asp-Arg and Asp-Cys. The method described in. The method according to any one of claims 1 to 5, wherein the sequence of the peptide includes the sequence of Ala-Thr-Pro-Glu-Asp-Asn-Gly-Arg-Ser-Phe-Ser. The method according to any one of claims 1 to 7, wherein the peptide is a lipopeptide or a glycopeptide. The peptide has the sequence Cys-Ala-Thr-Pro-Glu-Asp-Asn-Gly-Arg-Ser-Phe-Ser and has a side chain thiol group of Cys (2R) -2,3-. The method according to any one of claims 1 to 8, wherein the lipopeptide is substituted with a bis (palmitoyloxy) propyl group.

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